The present invention is directed to polysaccharides that can be used to induce the production of antibodies to specific strains of enterococcal bacteria. The polysaccharides may be incorporated into a vaccine or used in vitro to assay for the presence of bacteria in a sample of biological fluid.
Enterococci are frequently causes of serious infections in newborns and severely immunocompromised patients. Until recently, infections have been adequately controlled using antibiotics. However, drug-resistant bacterial strains are emerging, and infection by strains resistant to all presently available antibiotics may become a serious problem in the near future. It is therefore essential that new methods for controlling enterococcal infections be developed.
The present invention describes a procedure for purifying polysaccharides from the cell membranes of E. faecalis. When administered to an appropriate host, these antigens induce the production of high titers of opsonic antibodies that kill both E. faecalis and E. faecium. 
The present invention is based upon the discovery that a polysaccharide antigen can be isolated from E. faecalis bacteria and that this antigen can be used to produce antibodies that kill two different species of enterococci, E. faecalis and E. faecium. 
E. faecalis bacteria, preferably bacteria deposited as ATCC number 202159, are sequentially digested with mutanolysin/lysozyme, nucleases (both RNase and DNase) and pronase. The digestion product is then size fractionated and high molecular weight fractions containing the immunogenic polysaccharide are selected. The preferred method for carrying out the size fractionation step is to use a column of Sephacryl S-500(trademark) (Pharmacia, cross-linked allyl dextran/N,Nxe2x80x2-methylenebiscraylamide) and to collect fractions corresponding to the void volume.
Structural characterization of the high molecular weight polysaccharide thus obtained has demonstrated that it has a disaccharide, xcex1-D-GlcP-(1xe2x86x922)-xcex1-D-GlcP, linked to a glycerol teichoic acid backbone via the number 2 carbon of glycerol. There are phosphodiester bonds between the first carbon of the glycerol backbone and the sixth carbon of the glucose disaccharide. Substantially purified high molecular weight polysaccharides having these characteristics are part of the invention. As used herein, the term xe2x80x9csubstantially purifiedxe2x80x9d refers to polysaccharides that are essentially free from other biological materials. Typically the polysaccharides in such a preparation will constitute at least 80% of the total biological material present with higher percentages being preferred.
Once obtained, the high molecular weight polysaccharides may be incorporated as part of a pharmaceutically acceptable preparation and administered to an animal, e.g., a mouse, rabbit, sheep, goat etc., in order to generate antibodies that react with E. faecalis. Alternatively the high molecular weight polysaccharide can be incorporated into a vaccine and administered to people in order to evoke an immune response. As used herein, the term xe2x80x9cimmune responsexe2x80x9d means that the administration of vaccine confers upon the recipient protective immunity to subsequent challenge by E. faecalis bacteria.
Lower molecular weight polysaccharides may also be generated from the preparations described above. In order to destroy phosphodiester-linked materials, e.g., teichoic acid, the size fractionated material may be treated with hydrogen fluoride. The substantially purified low molecular weight polysaccharides thus obtained may be coupled to a protein or other suitable carrier and then used to generate antibodies or as vaccines in the same manner discussed above.
The high or low molecular weight polysaccharide-containing vaccines may be administered to patients at high risk for enterococci infection. Alternatively, they may be administered to a healthy individual for the purpose of developing antibodies that can be administered to an infected patient. Antibodies developed in a human or other species may also be used in assays to determine whether E. faecalis is present in a biological fluid. The present invention includes both the vaccines for immunizing patients as well as the immunization procedure itself.